The normal identity and function of a cell depend on accurate processing of its genetic code through successive cell divisions. This responsibility is handled by DNA metabolic proteins that replicate DNA and fix damage caused by replication errors or environmental insults. Inadequate processing of DNA can result in aberrant proteins that disrupt cellular function and lead to various disease states, including carcinogenesis. Among the variety of proteins that replicate and repair DNA, a few are conserved in structure/function in many organisms. These include DNA polymerase, and accessory proteins that help it function at high efficiency, such as the circular clamp that tethers polymerase to DNA during synthesis and the clamp loader that assembles the clamp around DNA for use by the polymerase. The principal investigator proposes to study the RFC clamp loader and PCNA clamp from the model eukaryote Saccharomyces cerevisiae. The 5-protein RFC complex functions as a machine-in that it uses energy from the chemical reaction of ATP binding/hydrolysis to perform the mechanical work of PCNA assembly on DNA. In order to understand precisely how this biological machine works, the proposed study seeks to answer questions such as: 1) How does RFC manipulate PCNA and DNA? 2) How does RFC bind and hydrolyze ATP? 3) What role do the multiple ATP-hydrolyzing RFC subunits play in the clamp assembly process? 4) How does RFC couple its ATPase activity to the work of placing PCNA onto DNA? Answers to these questions lie in the kinetic and thermodynamic parameters that govern RFC activity. The principal investigator will determine these parameters by quantitating the interactions between RFC and its substrates (ATP, DNA, and PCNA), as well as its ATPase and PCNA loading activities, using steady-state and transient-state kinetic techniques. Global analysis of the data describing the actions of RFC will reveal the detailed mechanism of how this clamp loader works in DNA metabolism. A flurry of recent reports indicates that RFC (and RFC-like clamp loaders) participate in cell cycle checkpoint controls, apoptosis, transcriptional silencing, and telomere-length regulation. The principal investigator anticipates that the proposed study of how RFC works will shed light on its activity in these critical cellular processes as well.